A process for making a detergent tablet by forming a disintegrating agent by compacting cellulose or a cellulose derivative, forming disintegrating agent particles comprising the disintegrating agent, dry mixing a washing- or cleaning-active substance and an amount of the disintegrating agent particles effective to rapidly dissolve or disperse the composition in water, and shaping the resulting mixture into a tablet. The disintegrating agent particles have a particle size distribution of less than 10% by weight smaller than about 0.2 mm particle size and no more than 1% by weight of dust-fine particles.

This invention relates to washing- or cleaning-active shapes, above all tablets, such as detergent tablets, dishwasher tablets, stain remover tablets or water softening tablets, for use in the home, more particularly for use in machines, to a process for the production of these shapes and to their use.
Washing- or cleaning-active shapes, more particularly tablets, have a number of advantages over powder-form compositions, including easy handling, simple dosing and low packaging volumes. However, problems arise out of the fact that comparatively high pressures have to be applied in the compression of the powder-form constituents in order to achieve adequate dimensional stability and fracture resistance. On account of the high compression involved, tablets of the type in question often show unsatisfactory disintegrating and dissolving properties in use so that the active substances are reduced too slowly in the washing or cleaning cycle and textiles in particular are in danger of being left with residues after the wash cycle.
The problem of the slow disintegration of tablets has been known for some time, more especially in the pharmaceutical industry. Here, the problem has been overcome or at least eased by the addition of certain disintegration aids known as tablet disintegrators. According to Rxc3x6mpp (9th Edition, Vol. 6, page 4440) and Voigt xe2x80x9cLehrbuch der pharmazeutischen Technologiexe2x80x9d (6th Edition, 1987), tablet disintegrators are auxiliaries which provide for the rapid disintegration of tablets in water or gastric juices and for the release of the pharmaceuticals in an absorbable form. According to their action mechanism, they are classed as substances which increase the porosity or capillarity (xe2x80x9cwick effectxe2x80x9d) of tablets and which have a high adsorption capacity for water, as gas-evolving substances for effervescent tablets or as hydrophilicizing agents which ensure that the constituent particles of tablets are wetted in water. The first class includes the substances known as traditional disintegrating agents, such as starch, cellulose and cellulose derivatives, alginates, dextrans, crosslinked polyvinyl pyrrolidones and many others while the second class includes systems of weak acids and carbonate-containing agents, more particularly citric acid and tartaric acid in combination with hydrogen carbonate or carbonate. Examples of the third class are polyethylene glycol sorbitan fatty acid esters.
Thus, it is proposed in German patent application 938 566 to convert acetyl salicylic acid before compression into granules and, after complete but careful drying, to coat the granules thus formed with highly disperse silica. The acetyl salicylic acid granules coated with the highly disperse silica powder may then be mixed with other tablet ingredients, which may be present in powder form or granular form, and the resulting mixture is tabletted. The separating layer of highly disperse silica not only acts as an insulating layer and as protection against unwanted reactions, it also contributes towards the rapid disintegration of the tablets, even after prolonged storage.
German patent application 12 28 029 describes a process for the production of tablets in which powder mixturesxe2x80x94without preliminary granulationxe2x80x94are first mixed with cellulose powder and optionally highly disperse silica and the resulting mixture is compressed, in one preferred embodiment after grinding.
According to German patent application 41 21 127, a particularly effective auxiliary in the production of pharmaceutical tablets contains cellulose particles with a coating material fixed to their surface. The auxiliary is used in the form of fine particles, mean particle sizes below 200 xcexcm being described as particularly advantageous. These fine-particle auxiliaries, whichxe2x80x94in the production of pharmaceuticalsxe2x80x94lead to tablets with both a relatively high fracture resistance and a relatively high disintegration rate, are produced in particular by a grinding process carried out in a ball mill.
Accordingly, conventional tablet disintegrators belonging to the first class mentioned above are normally either mixed in very fine-particle form with the other tablet ingredients, which may be present in the form of fine particles or granules, before compression or the other tablet ingredients are coated or powdered/dusted with the tablet disintegrator.
According to the teaching of European patent EP-B 0 523 099, disintegrators known from the production of pharmaceuticals may also be used in detergents or cleaning products. The disintegrators mentioned include swellable layer silicates, such as bentonites, natural materials and derivatives thereof based on starch and cellulose, alginates and the like, potato starch, methyl cellulose and/or hydroxypropyl cellulose. These disintegrators may be mixed with, or even incorporated in, the granules to be compressed.
According to International patent application WO-A-96/06156 also, it can be of advantage to incorporate disintegrators in detergent or dishwasher tablets. Once again, microcrystalline cellulose, sugars, such as sorbitol, and also layer silicates, more particularly fine-particle swellable layer silicates of the bentonite and smectite type, are mentioned as typical disintegrators. Substances which contribute towards gas formation, such as citric acid, bisulfate, bicarbonate, carbonate and percarbonate, are also mentioned as possible disintegration aids.
Although neither of the last two prior-art documents cited above specifies the exact particle size distribution which suitable disintegrators are supposed to have, figures relating to the microcrystallinity of the cellulose and the particle fineness of the layer silicates suggest to the expert, above all in connection with the literature known from the production of pharmaceutical tablets, that conventional disintegrators are supposed to be used in fine-particle form. This is consistent with the fact that, hitherto, relatively coarse products obtained, for example, by granulation of fine powders, which are expressly marketed as tablet disintegrators, have not been commercially available.
European patent applications EP-A0 466 485, EP-A-0 522 766, EP-A-0 711 827, EP-A-0 711 828 and EP-A-0 716 144 describe the production of cleaning-active tablets in which compacted particulate material with a particle size of 180 to 2000 xcexcm is used. The resulting tablets may have both a homogeneous structure and a heterogeneous structure. According to EP-A-0 522 766, the surfactant- and builder-containing particles at least are coated with a solution or dispersion of a binder/disintegration aid, more particularly polyethylene glycol. Other binders/disintegration aids are the already repeatedly described and known disintegrating agents, for example starches and starch derivatives, commercially available cellulose derivatives, such as crosslinked and modified cellulose, microcrystalline cellulose fibers, crosslinked polyvinyl pyrrolidones, layer silicates, etc. Other suitable coating materials are weak acids, such as citric acid or tartaric acid which, in conjunction with carbonate-containing sources, lead to effervescent effects on contact with water and which, according to Rxc3x6mpp""s definition, belong to the second class of disintegrating agents. In these cases, too, no specific details are provided as to the particle size distribution of the disintegrators. However, they are all applied to the surface of granules. This is done eitherxe2x80x94as mentionedxe2x80x94in liquid to disperse form or in solid form. It is known to the expert in this connection that fine-particle solids, i.e. powder-like solids, which normally also contain relatively high percentages of dust, can be used for coating particles with particulate solids, so-called xe2x80x9cpowderingxe2x80x9d.
According to EP-A-0 711 827, the use of particles consisting predominantly of citrate, which has a certain solubility in water, also leads as a secondary effect to accelerated disintegration of the tablets. It is assumed that the dissolution of the citrate locally increases the ion strength over a transitional period so that the gelling of surfactants is suppressed and, as a result, the disintegration of the tablet is not impeded. According to this patent application, therefore, citrate is not a disintegrating agent in the accepted sense, but acts as an anti-gelling agent.
The proposed solutions mentioned in the foregoing produce the required result in the production of pharmaceutical tablets. Although, in the field of detergents and cleaning products, they contribute towards an improvement in the disintegration properties of washing- or cleaning-active tablets, the improvement achieved is inadequate in many cases. This applies in particular when the percentage of tacky organic substances in the tablets, for example anionic and/or nonionic surfactants, increases. This is one of the reasons why, hitherto, detergent tablets which satisfy stringent consumer requirements have not been commercially available. However, in the field of dishwashing detergents and detergent additives also, tablets do not have a sufficiently high disintegration rate despite an often satisfactory fracture resistance. Increasing the rate at which dishwasher tablets also disintegrate and dissolve can have advantages, particularly for phases which contain active substances that are supposed to be effective at the beginning of the dishwashing process or at relatively low temperatures.
Accordingly, the problem addressed by the present invention was to provide washing- or cleaning-active shapes which would contain a disintegrating agent with a high adsorption capacity for water that would be capable of increasing the porosity or the capillarity of the tablets and which would not have any of the disadvantages mentioned above. Another problem addressed by the invention was to provide a process for the production of these improved washing- or cleaning-active shapes.
It has now been found that the conventional disintegrating agents known from the production of pharmaceutical tablets lead to rapidly disintegrating washing- or cleaning-active shapes providing they are not used in the usual way.
In a first embodiment, therefore, the present invention relates to a washing- or cleaning-active shape containing at least one disintegrating agent which is capable of increasing the porosity or capillarity of shapes, more particularly tablets, and which has a high adsorption capacity for water, this disintegrating agent being present in the shape in granular and optionally in co-granulated form, the granules of disintegrating agent (disintegrator granules) containing at least 20% by weight and preferably 25 to 100% by weight of the disintegrating agent orxe2x80x94where several disintegrating agents are usedxe2x80x94the disintegrating agents and the particle size distribution (sieve analysis) being such that at most 1% by weight, preferably less, of dust-fine particles are present and a totalxe2x80x94including any dust-fine particles presentxe2x80x94of less than 10% by weight of the disintegrator granules being smaller than 0.2 mm in size. In one advantageous embodiment, at least 90% by weight of the disintegrator granules have a particle size of at least 0.2 mm and at most 3 mm.
In the context of the present invention, disintegrating agents in granular form or in co-granulated form or disintegrator granules are understood to include any disintegrating agents which are present per se in the form of fine-particle powders and which have been converted into coarser particles by spray drying, granulation, agglomeration, compacting, pelleting or extrusion.
A definition of what is meant by washing- or cleaning-active shapes was given earlier on. They are primarily cylindrical objects or tablets which may be used as laundry detergents, dishwashing detergents, bleaching agents (spotting salts) and optionally as pretreatment agents, for example as water softeners or bleaching agents. However, the term xe2x80x9cshapexe2x80x9d is not confined to tablets and, in principle, encompasses any three-dimensional form which the starting materials can be made to assume, optionally under the effect of an external container. Cylindrical shapes can have a height which is smaller or greater than or equal to the diameter of the base. However, the shapes may also have an angular base, for example a rectangular base, more particularly a square base, or even a rhombic or trapezoidal base. Other versions include three-cornered or more than four-cornered bases of the shape.
By virtue of the outstanding disintegrating properties of the shapes according to the invention, it is possible, but not absolutely essential, directly to introduce the shapes into the aqueous liquor of a machine washing process by means of a dispenser. It is even possible to place the shape or shapes in the dispensing compartment of commercially available domestic machines, more particularly washing machines. Accordingly, in one preferred embodiment of the invention, the three-dimensional form of the shapes is adapted in its dimensions to the dispensing compartment of commercially available domestic machines.
Another preferred shape has a plate-like or slab-like structure with alternately thick long segments and thin short segments, so that individual segments can be broken off from this xe2x80x9cbarxe2x80x9d at the predetermined weak spots, which the short thin segments represent, and introduced into the machine or rather into the dispensing compartment of the machine. This xe2x80x9cbarxe2x80x9d principle can also be embodied in other geometric forms, for example vertical triangles which are only joined to one another at one of their longitudinal sides.
In one preferred embodiment, the invention provides homogeneous or heterogeneous shapes, more particularly tablets, the tablets having a diameter of preferably 20 to 60 mm and, more preferably, of 40xc2x110 mm. The height of these tablets is preferably 10 to 30 mm and more preferably 15 to 25 mm. The weight of the individual shapes, more particularly the tablets, is preferably between 15 and 60 g and more preferably between 25 and 40 g per shape or tablet. By contrast, the density of the shapes or tablets normally assumes values above 1 kg/dm3 and preferably between 1.1 and 1.4 kg/dm2. Depending on the nature of the application, the water hardness range and the degree of soiling, one or more shapes, for example 2 to 4 shapes, more particularly tablets, may be used. Other shapes according to the invention may even have smaller diameters or dimensions, for example of the order of 10 mm.
A homogeneous shape in the context of the present invention is one in which the ingredients of the detergent are uniformly distributed throughout the shape. Accordingly, heterogeneous shapes are shapes in which the ingredients are not homogeneously distributed. Heterogeneous shapes may be produced, for example, by compressing the various ingredients to form a shape comprising several layers, i.e. at least two layers, rather than into a monolayer shape. These various layers may have different disintegration and dissolving rates. This can provide the shapes with favorable performance properties. If, for example, the shapes contain ingredients which adversely affect one another, one component may be integrated in the more quickly disintegrating and dissolving layer while the other component may be incorporated in a more slowly disintegrating layer so that the first component can act in advance or can already have reacted off by the time the second component dissolves. The various layers of the shapes can be arranged in the form of a stack, in which case the inner layer(s) dissolve at the edges of the shape before the outer layers have completely dissolved or disintegrated. Alternatively, however, the inner layer(s) may also be completely surrounded by the layers lying further to the outside which prevents constituents of the inner layer(s) from dissolving prematurely.
In another preferred embodiment of the invention, a tablet consists of at least three layers, i.e. two outer layers and at least one inner layer, a peroxy bleaching agent being present in at least one of the inner layers whereas, in the case of the stack-like tablet, the two cover layers and, in the case of the envelope-like tablet, the outermost layers are free from peroxy bleaching agent. In another possible embodiment, peroxy bleaching agent and any bleach activators or bleach catalysts present and/or enzymes may be spatially separated from one another in one and the same tablet/shape. Embodiments such as these have the advantage that, even in cases where the shape/tablet of detergent or bleaching agent is introduced into the washing machine or into the hand washing bowl in direct contact with the fabrics, there would be no danger of spotting by bleaching agent or the like on the fabrics.
Other examples of heterogeneous shapes can be found, for example, in European patent applications EP-A-0 711 827, EP-A-0 711 828 and EP-A-0 716 144.
According to the above definition, several disintegrating agents may be used either individually or in combination, being present in the same disintegrator granules or in various disintegrator granules. Where various disintegrator granules are to be used, preferably more than 40% by weight, more preferably at least 50% by weight and, most preferably, at least 60% by weight, based on the total quantity of disintegrator granules used, have a composition and particle size distribution of the type mentioned above. However, since it is precisely the coarser than usual type of disintegrating agent used which accelerates disintegration of the washing- or cleaning-shape, it is of particular advantage, and highly desirable, for all the various disintegrator agent granules used to have the features mentioned above.
Preferred disintegrating agents which have to be converted into granular form or into co-granulated form include starch and starch derivatives, cellulose and cellulose derivatives, for example microcrystalling cellulose, CMC, MC, alginic acid and salts thereof, carboxymethyl amylopectin, polyacrylic acid, polyvinyl pyrrolidone and polyvinyl polypyrrolidone. The disintegrator granules may be conventionally produced, for example by spray drying or superheated steam drying of aqueous preparations or by granulation, pelleting, extrusion or roll compacting. It can be of advantage to incorporate additives, granulation aids, carriers or coating agents of known types in the disintegrating agents (co-granulated form). In one preferred embodiment of the invention, additives are non-surface-active ingredients of detergents or cleaning compositions, more particularly bleach activators and/or bleach catalysts. Particularly preferred disintegrating agent granules are those which contain tetraacetyl ethylenediamine (TAED) and/or other conventional bleach activators as additives. Disintegrator granules such as these may advantageously be produced by co-granulation of the disintegrating agent with the additive. co-granulation in this way can increase the distribution of the disintegrating agent in the shape, more especially in the tablet, which in certain cases can also lead to an improvement in the disintegration rate of the shape/tablet.
According to the present invention, it is particularly preferred to use cellulose-containing disintegrating agents of the type described in earlier German patent application 197 09 991.2. These disintegrating agents are cellulose-containing materials which have been compacted, compacted wood-based materials, such as TMP (thermomechanical pulp) or CTMP (chemothermomechanical pulp) preferably being used. Particularly preferred disintegrating agents such as these are commercially available from the Rettenmaier Company, for example, under the names of Arbocel(copyright) B and Arbocel(copyright) BC (beech cellulose), Arbocel(copyright) BE (beech sulfite cellulose), Arbocel(copyright) B-SCH (cotton cellulose), Arbocel(copyright) FIC (spruce cellulose) and other Arbocel(copyright) types (Arbocel(copyright) TF-30-HG).
In one embodiment of the invention, the content of actual disintegrating agents in the disintegrator granules is preferably from 50 to 100% by weight and more preferably at least 70% by weight, embodiments containing at least 80 or even 90% by weight or more being particularly advantageous. Disintegrator granules which are made almost entirely of the commercially available disintegrating agents, i.e. contain between 97 and 100% by weight of the commercially available disintegrating agents, are also possible.
In another preferred embodiment of the invention in which the disintegrating agent is used in co-granulated form, more especially in combination with TAED, in the disintegrator granules, the content of disintegrating agent in the granules is more than 20% by weight and less than 70% by weight, advantageously at least 70% by weight and more particularly from 80 to 100% by weight of the other constituents, based on the other constituents in the disintegrator granules, consisting of active substances, such as bleach activator, more especially TAED, and/or bleach catalyst.
If fines smaller than 0.2 mm in size should accumulate in the production of the disintegrator granules, not only is it preferred to remove them to such an extent that the disintegrator granules are substantially free from dust (dust in the context of the invention being particles smaller than 0.1 mm in size, see above), but also to such an extent that the total content of particles smaller than 0.2 mm in size is minimized to 0-5% by weight. In another preferred embodiment, at least 90% by weight of the disintegrator granules have a particle size of at least 0.3 mm and at most 3 mm, more particularly up to at most 2 mm.
In one preferred embodiment, the shapes according to the invention contain disintegrator granules in quantities of 1 to 20% by weight and preferably in quantities of 2 to 15% by weight, quantities of up to 10% by weight being particularly preferred.
In another preferred embodiment of the invention, not only the disintegrator granules, but also the other constituents of the detergent shape are mainly present in the particulate form mentioned in the foregoing. Thus, preferably at least 50% by weight of the other constituents and more preferably at least 70% by weight have a particle size distribution of 0.2 to 3 mm. In this case, too, it is particularly important that the other constituents should only contain 0 to 5% by weight of particles smaller than 0.2 mm in size. In one particularly advantageous embodiment, at least 90% by weight of the other constituents have particle sizes of 0.2 to 3.0 mm. In the other constituents also, dust should be avoided as far as possible. This is achieved, for example, by the other constituents being present in granular form and/or being combined in one or more compounds which may be conventionally produced, for example by spray drying, superheated steam drying, granulation/agglomeration, fluidized bed granulation, roll compacting, pelleting or extrusion. Any fines smaller than 0.2 mm in size accumulating in the production of these compounds are preferably removed before mixing with the disintegrator granules. Surface treatment compositions, such as powdering agents, which are known to consist of very fine particles and which are just not used in coarse-particle form, are specifically not included in the overall particle size distribution of the other constituents. Both disintegrator granules and also other constituents may be aftertreated with solid fine-particle surface treatment compositions.
The other constituents may be any typical detergent ingredients, pretreatment compositions, bleaching agents and water softeners. These include above all anionic, nonionic, cationic, amphoteric and zwitterionic surfactants, inorganic and organic, water-soluble or water-insoluble builders and co-builders, bleaching agents, more especially peroxy bleaching agents, and active chlorine compounds which are advantageously coated, bleach activators and bleach catalysts, enzymes and enzyme stabilizers, foam inhibitors, redeposition inhibitors, substances which prevent the resoiling of fabrics, so-called soil repellents, and typical inorganic salts, such as sulfates and organic sulfates, such as phosphonates, optical brighteners and dyes and perfumes. In addition, the use of conventional silver protectors is recommended for machine dishwashing detergents.
Preferred anionic surfactants include both those based on petrochemicals, such as alkyl benzenesulfonates and alkane sulfonates and alkyl (ether)sulfates with odd chain lengths, and those based on native materials, for example fatty alkyl sulfates or fatty alkyl (ether)sulfates, soaps, sulfosuccinates, etc. Alkyl benzenesulfonates and/or various chain cuts of alkyl sulfates or alkyl ether sulfates are particularly preferred, optionally in combination with small quantities of soap. Whereas, in the case of alkyl benzenesulfonates, C11-13 alkyl benzenesulfonate and C12 alkyl benzenesulfonate are preferred, preferred chain cuts in the case of the alkyl (ether)sulfates are C12 to C16, C12 to C14, C14 to C16, C16 to C18 or C11 to C15 or C13 to C15.
Preferred nonionic surfactants include in particular C12-18 fatty alcohols ethoxylated with on average 1 to 7 moles of EO per mole of alcohol and the corresponding C11-17 alcohols, more particularly C13-15 alcohols, and the more highly ethoxylated alcohols with the chain lengths mentioned known from the field of detergents and cleaning compositions, amine oxides, alkyl polyglycosides, polyhydroxyfatty acid amides, fatty acid methyl ester ethoxylates and gemini surfactants.
Preferred inorganic builders are, in particular, conventional phosphates, preferably tripolyphosphate, zeolites, more especially zeolite A, zeolite P, zeolite X and mixtures thereof, and carbonates, hydrogen carbonates and crystalline and amorphous silicates with multiple wash cycle performance. Conventional co-builders include, above all, (co)polymeric salts of (poly)carboxylic acids, for example copolymers of acrylic acid and maleic acid, and also polycarboxylic acids and salts thereof, such as citric acid, tartaric acid, glutaric acid, succinic acid, polyaspartic acid, etc. The expert knows the organic co-builders suitable for use in accordance with the invention from innumerable publications on the subject of detergents and cleaners.
Suitable bleaching agents are, above all, the peroxy bleaching agents widely used at the present time, such as perborate and percarbonate, above all in combination with conventional bleach activators and bleach catalysts, more especially in the field of dishwashing detergents, and the active chlorine compounds mentioned earlier on.
Among the enzymes, not only proteases, but also lipases, amylases, cellulases and peroxidases and combinations of these enzymes are of particular interest.
A preferred embodiment of the invention is characterized by the use of anionic surfactant-containing compounds in various anionic surfactantsxe2x80x94for example alkyl sulfates and alkenyl benzenesulfonates and/or soap or even alkyl sulfates and sulfonated fatty acid glycerol estersxe2x80x94and/or anionic surfactants are present in combination with nonionic surfactants, for example alkyl sulfates of various chain lengths, optionally even several types of alkyl sulfates with various chain lengths in combination with ethoxylated alcohols and/or other nonionic surfactants of the type mentioned above. For example, anionic and nonionic surfactants may also be predominantly accommodated in two different compounds.
In another preferred embodiment of the invention, at least 50% by weight and preferably 60 to 100% by weight of the other constituents are aftertreated before mixing with the disintegrator granules, i.e. are sprayed or powdered under granulating conditions, the water-free aftertreatment being particularly preferred. Preferred liquid constituents include nonionic surfactants and/or polyethylene glycols. However, in another particularly preferred embodiment, the other constituents may also be aftertreated with a water-free melt of nonionic compounds solid at room temperature, more particularly with polyethylene glycols having relative molecular weights above 2,000 and above all between 4,000 and 12,000. As in the case of the disintegrator granules, suitable powdering agents are, above all, fine-particle zeolites, silicas, sulfates, calcium stearates, phosphates and/or acetates. In another preferred embodiment of the invention, dust and particles smaller than 0.2 mm in size are completely removed before mixing with the disintegrator granules. Applicants assume that this known measure of surface treatment delays dissolution of the particles in the shape/tablet before its actual disintegration and, for this reason, contributes towards the particularly outstanding disintegration properties of the shapes/tablets in the aqueous liquor in the production of shapes in combination with the disintegrator agent granules having a specific particle size distribution.
The invention can also make use of the fact that acidifying agents, such as citric acid, tartaric acid or succinic acid, and also acidic salts of inorganic acids (xe2x80x9chydrogen saltsxe2x80x9d), for example bisulfates, above all in combination with carbonate-containing systems, can also contribute towards improving the disintegration properties of the shapes. According to the invention, however, these acidifying agents are also used in the form of coarse particles, more particularly granules, which are substantially free from dust and which are adapted in their particle size distribution to the disintegrator granules. The granular acidifying agents may be present in the shapes, for example, in quantities of 1 to 10% by weight.
As already repeatedly mentioned, the shapes according to the invention, more especially the hitherto poorly disintegrating and poorly soluble detergent tablets and bleach tablets, have outstanding disintegration properties. This can be tested, for example, under critical conditions in a normal domestic washing machine bleach/detergent tablet used directly in the wash liquor with the aid of a conventional dispenser, delicates program or coloreds program, washing temperature max. 40xc2x0 C.) or in a glass beaker at a water temperature of 25xc2x0 C. The carrying out of the corresponding tests is described in the Examples. Under these conditions, the shapes according to the invention not only disintegrate completely in 10 minutes, the preferred embodiments have disintegration times in the glass beaker test of less than 3 minutes and, more particularly, less than 2 minutes. Particularly advantageous embodiments even have disintegration times of less than 1 minute. Disintegration times of less than 3 minutes in the glass beaker test are sufficient to ensure than the detergent shapes or detergent additive shapes are flushed into the wash liquor from the dispensing compartment of conventional domestic washing machines. In another embodiment, therefore, the present invention relates to washing process in which the detergent shapes are introduced into the wash liquor from the dispensing compartment of a domestic washing machine. The dissolving time of the detergent shapes in the washing machine is preferably less than 8 minutes and more preferably less than 5 minutes.
The actual production of the shapes according to the invention is carried out by initially dry mixing the disintegrator granules with the other constituents and then shaping the resulting mixture, more particularly by compression, into tablets using conventional processes (for example as described in the conventional patent literature on tabletting, above all in the field of detergents of cleaners, more particularly as described in the above-cited patent applications and in the article entitled xe2x80x9cTablettierung: Stand der Technikxe2x80x9d in Sxc3x96FW Journal, Vol. 122, pages 1016-1021 (1996).

Process for preparing household detergent or cleaner shapes


The present specification discloses a blockchain computer application. This blockchain computer application includes a Graphical User Interface (GUI) that supports the organization and blockchaining of files to a blockchain. This GUI includes a virtual blockchain organizational tool onto which users can drag and drop files and organize them into various virtual blockchain blocks without permanently attaching those files to an actual blockchain until the virtual blockchain is converted to an actual blockchain. The conversion process preserves the organizational structure of the files set within the virtual blockchain. Files may also be automatically blockchained to a specific blockchain by dragging them and dropping them onto a blockchain application desktop icon. Files may also be automatically blockchained to specific blockchains by dragging them and dropping them onto a blockchain icon within the GUI where each blockchain icon is directly associated with a separate blockchain.

A blockchain is a continuously growing list of records, called blocks, which are linked and secured using cryptography. Each block typically contains a cryptographic hash of the previous block, a timestamp, and transaction data. By design, a blockchain is inherently resistant to modification of the data. It is an open, distributed ledger that can record transactions between two parties efficiently and in a verifiable and permanent way. For use as a distributed ledger, a blockchain is typically managed by a peer-to-peer network collectively adhering to a protocol for validating new blocks. Once recorded, the data in any given block cannot be altered retroactively without the alteration of all subsequent blocks, which requires collusion of the network majority. Blockchains are secure by design and are an example of a distributed computing system with high Byzantine fault tolerance. Decentralized consensus has therefore, been achieved with a blockchain. This makes blockchains potentially suitable for the recording of events, medical records, and other records management activities, such as identity management, transaction processing, documenting provenance, food traceability, or voting. The first work on a cryptographically secured chain of blocks was described in 1991 by Stuart Haber and W. Scott Stornetta. In 1992, Bayer, Haber, and Stornetta incorporated Merkle trees to the design, which improved its efficiency by allowing several documents to be collected into one block.

Computer application for blockchaining files through a graphical user interface


The present invention provides a method for carrying out in vitro the complete developmental sequence culture of tissular parasites, which includes culturing the parasites in a totally defined culture medium which is an axenic monophasic liquid culture medium, free of serum and free of serum-derived or cell-derived macromolecules, proteins and peptides. For obtaining amastigote forms, this medium is buffered at a pH of 5.5 to 6.5 and has an osmolarity of at least 400 milliosmoles/kg of liquid. For obtaining promastigote forms, the medium is buffered at a pH of 7 to 7.5 and has an osmolarity of at least 300 milliosmoles/kg liquid. Application to the in vitro culture of different stages of tissular parasites such as Leishmania, T. cruzi, and hamatoprotozoa is also provided.

A subject of the invention relates to a process for the in vitro culture of different stages of the developmental cycle of a parasite. It also relates to the parasitic forms obtained and their biological uses.
By culture is meant, in the description which follows and the claims, both the adaptation of the parasitic form by successive passages in a given medium, and complete differentiation when it occurs for the adaptation and the culture itself of the parasitic form.
It is known that parasites constitute a real plague causing, by the intermediary of vectors, the infection of millions of people and animals.
Thus, leishmaniases, which are widespread throughout the world, are caused by protozoan of the Leishmania genus which are usually transmitted by a sand fly, Phlebotomus. Leishmaniases of the Old World and those of the New World are usually distinguished according to their geographical localization. They have very diverse clinical forms which differ significantly by their seriousness and their effect on health. Cutaneous, mucocutaneous (attacking nasal, buccal mucous and those of the ears) and visceral leishmaniases are distinguished.
As another parasite having a devastating effect, there can also be mentioned Trypanosoma cruzi, responsible for Chagas"" disease. In South America, it causes the infection of millions of people. Over 150 species of wild and domestic animals can be counted which can serve as hosts to the parasite which is transmitted to man by a bug, namely a Triatoma, which feeds on blood.
The infection can pass unnoticed for several years until the trypanosomas attack the nervous system, the heart or the digestive system.
The development cycle of many parasites includes various parasitic stages. That of Leishmania, for example, includes two stages having important differences at the structural, morphological, biochemical, immunological and physiological level, namely
in a vector insect, a flagellated form, called promastigote, which multiplies by scissiparity before acquiring its infectious form, for the mammalian host, also called metacyclic,
in a mammalian host, a non-flagellated stage, called amastigote, which exclusively parasitizes mononuclear phagocyte cells.
The differentiation into amastigotes occurs after attachment and penetration of the promastigotes into the monocytes. But only the amastigote forms persist and multiply inside the phagolysosome of the macrophages of the infected host.
In T. cruzi, this cycle includes three different parasitic stages, an epimastigote form, multiplication form of the vector insect, and the amastigote and trypomastigote forms, which are present in the infected host.
At present, most of the research carried out on the diagnosis of these parasitoses and the development of vaccines is conducted on the promastigote form whose production in culture is easy and quick.
Now, the only form present in the infected host is the amastigote form which, by persisting throughout the infection, triggers the immune response and participates in the development of the pathology. The danger of systematically extrapolating the experimental results obtained with the cultured promastigote forms within the scope of immuno-prophylactic, diagnostic or therapeutic studies aimed at the amastigote forms will be well understood.
In order to respond to this problem, various authors have been interested in obtaining amastigote forms. Thus the obtaining of amastigotes from tissues of experimentally infected animals or from cultures of infected macrophages have been reported. But this involves long and tedious isolation methods, which moreover lead to a limited number of parasites being obtained, which are often degenrated and which are incapable of multiplying and of surviving for longer than 2 to 3 days.
Furthermore, such amastigote forms are contaminated with cells, fragments and molecules derived from the macrophages, tissues or plasma of the host, designated hereafter by xe2x80x9ccellular contaminantsxe2x80x9d, limiting or making impossible the realization of many studies.
Culturing processes under axenic conditions, that is to say in the absence of any cellular contaminant, have been proposed for some species of leishmanias and for T. cruzi, but they do not allow an abundant and continual source of amastigote forms to be made available, and do not appear to be generally applicable to a large number of species and to different germs.
An aim of the invention is to resolve the above disadvantages and to provide more satisfactory experimental models by producing the desired parasitic stages in specific culture media, of totally defined simplified composition.
The invention relates in particular to providing a process generally applicable to a large number of species of a given parasite, allowing homogeneous populations of a given parasitic stage to be produced in a continuous manner and in unlimited quantities.
It relates more particularly to a process allowing these stages to be obtained in a form which is free from any cellular and seric contaminant, having the characteristic of not containing any macromolecules. By macromolecules is meant, in the description and the claims which follow, non-dialyzable molecules with a cut-off threshold of 3 kDa, that is to say having an apparent molecular weight of greater than 3 kDa (for example seric protein such as albumin).
It also relates to the in vitro production of the complete development cycle of parasites under axenic and aseric conditions.
According to another aspect, the invention relates to the new parasitic forms obtained, corresponding to the different stages of the parasitic cycle and, for each stage, to the different phases of their growth.
According to another aspect, the invention relates to the applications of the parasitic stages obtained and of the products produced or isolated from these stages, in particular in the domain of the diagnosis of parasitoses, immunoprophylaxis and screening of drug activities.
The process according to the invention, for the in vitro culture of different stages of tissular parasites, such as leishmania, T. cruzi, or also hematoprotozoa is characterized in that it is carried out in an axenic and aseric, liquid single-phase culture medium, free from macromolecules (non-dialyzable at a cut-off threshold of 3 kDa) and that for obtaining amastigote forms, this medium is buffered at a pH of 5.5 to 6.5 and has an osmolarity of at least 400 milliosmoles/kg of liquid, and in particular from 400 to 550 milliosmoles/kg of liquid, or that, for obtaining promastigote forms, this medium is buffered at a pH of 7 to 7.5 and has an osmolarity of at least 300 milliosmoles/kg of liquid, and in particular from 300 to 380 milliosmoles/kg of liquid.
The pH value of these media, within the range indicated above, ensures that the culture conditions are strictly controlled.
In the case of the culture of amastigotes, when the pH is greater than 6.5, a tendency towards the retransformation of the amastigotes into promastigotes is in fact noted and when it is less than 5.5, a poor growth is observed.
According to a preferred method of the invention, for obtaining amastigote forms, a culture medium is used containing a base medium, produced essentially from:
at least one culture medium for insect cells which has added to it inorganic salts, of Hanks"" salts type,
products which are sources of amino acids, such as L-glutamine and soja bean extracts,
sugars, such as D-glucose.
As soja bean extact, there can advantageously be used that marketed under the Trade mark trypto casein soja(copyright).
The culture medium for insect cells is advantageously the medium 199 H marketed by GIBCO.
Different compositions of this 199 HR medium are given in the GIBCO BRL catalogue page 48, 1992 edition.
A specially preferred composition for the production of base media carries the reference 042-01181 on page 48 of this catalogue, 1992 edition. The 199H M medium is more especially used, to which NaHCO3 and L-glutamine are added.
This medium, to which the above compounds are added, is advantageously buffered, for example with a buffer such as HEPES.
A preferred composition of the base medium contains several culture media for insect cells.
A base medium of this type results from the addition to the 199 H medium as marketed by Gibco, to which the compounds mentioned above have been added, of another medium such as the modified 199 H medium as marketed by Flow. A composition of this medium is given in the Flow Laboratories catalogue, 1992 under the reference 14230-54. Before adding it to the initial preparation, this latter modified 199 H medium is subjected to a thermal treatment.
The base media defined above are new and, as such, are also a subject of the invention. They can be stored for several months at xe2x88x9220xc2x0 C.
In order to avoid oxidation of the parasitic stages, this base medium has added to it, at the time of use, anti-oxidizing agents such as hemin, which also has the advantage of constituting a source of iron and agents with a reducing effect such as reduced glutathionee. Vitamins are also advantageously added. A suitable mixture of vitamins includes biotin, calcium D pantothenate, choline chloride, folic acid, inositol, nicotinamide, para-aminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, some of which are used and advantageously all of which are used.
The resultant culture media are advantageously characterized in that they are free from nucleotides as additives. They can be stored at +4xc2x0 C. for about two weeks without alteration to their properties.
The use of the resultant culture media, as illustrated by the examples given hereafter, allows amastigote forms to be produced which are capable of mass multiplication in vitro, in a continuous manner.
The axenic and aseric media have the advantage of being free from all macromolecules and in particular from those present in foetal calf serum and/or originating from the host cells, which can mask other constituents (seric and cellular proteins).
These media are particularly suitable for the amastigote forms of tissular protozoa, such as cutaneous or muco-cutaneous or visceral leishmanias, or various clones of T. cruzi, or also Plasmodium or Babesia.
As species of leishmanias, there can be mentioned L. mexicana, L. amazonensis, and L. braziliensis or also L. major, L. guyanensis and L. panamensis. Other preferred culture media, particularly suitable for the culture of the amastigote forms of visceral leishmanias, such as L. donovani, L. infantun and L. chagasi, contain, in addition, sulphurous compounds. These are in particular sulphurated amino acids, such as cysteine, and notably the L form, and/or nutritive products such as bathocuproine sulphonic acid.
For the differentiation of promastigote forms into amastigote forms, in the case of Leishmania, the cellular culture medium represents, relative to the final medium, about 8 to 15% (V/V), notably of the order of 10%, the amino acids, or products which are sources of these amino acids, such as soja trypto casein R and L-glutamine, are present at a rate of about 4 to 8% (W/V), notably about 5 to 6%, the supply of sugar, notably as glucose, is advantageously carried out at a rate of about 2 to 4% (W/V), notably 2 to 3%, the anti-oxidizing agents such as hemin, at a rate of 0.0002 to 0.0015% (w/V), notably of the order of 0.0005%, and glutathione at a rate of 0.01% to 0.05%, notably of the order of 0.025% and the vitamin solution (100xc3x97) at a rate of 1% to 5% (V/V), notably of the order of 2%.
The sulphurous compounds, when they are used, in particular L-cysteine, are used at a rate of about 0.25 to 0.50% (W/V), notably of the order of 0.3%, and bathocuproine sulphonic acid is used at a rate of about 0.004 to 0.008% (W/V), notably of the order of 0.005%.
For the culture of the promastigote stages, a culture medium is advantageously used which is produced from a medium suitable for cell culture, such as RPMI 1640 medium, to which are added amino acids such as L-glutamine and a buffer to adjust the pH to a value of 7 to 7.5, this medium also having added to it another medium suitable for cell culture, in particular 199H M medium, containing inorganic salts such as Hanks"" salts, and anti-oxidizing agents, such as hemin.
This medium is therefore free from any seric contaminant and contains no macromolecule as proved by 10% polyacrylamide gel analysis.
The 199H M medium is used at a rate of about at least 2% (V/V), notably about 2 to 10% and bovine hemin is used at a rate of 0.0001 to 0.0015% (W/V), notably of the order of 0.0005%.
The simplicity of preparation of such a medium from products which are already being marketed will be observed. Moreover the absence of serum advantageously produces an inexpensive product.
In accordance with the invention, these culture media are used in a process for the continuous mass production of parasitic forms.
For the adaptation and continuous culture in vitro of amastigote forms, a suitable culture medium as defined above is inoculated with promastigotes at the end of the exponential phase, at a rate of 106 to 107 promastigotes/ml of medium.
The conditions for carrying out the adaptation and culture are advantageously chosen in such a way as to ensure a total transformation of the parasites in a reproducible manner.
The adaptation, then the culture, are carried out at temperatures of the order of 28 to 36xc2x0 C., and notably around 32xc2x0 C. at a pH of 5.5 to 6.5. Usually a transformation of the promastigote forms into amastigote forms in excess of 90% is observed in 6 to 7 days. This transformation is for example total in 4 days for the Leishmania, after a number of passages which varies according to the species studied and which generally corresponds to 3 to 9 subcultures, and which decreases when the incubation temperature increases.
For the adaptation and continuous culture in vitro of so-called primary culture or short-term promastigote forms of parasites, such as Leishmania, directly obtained from the amastigote forms, inoculation with the amastigote forms as obtained above is carried out, at a rate of 106 to 107 amastigote forms/ml of medium. Amastigote forms at the end of the exponential phase are advantageously used. The culture is carried out at a temperature close to ambient temperature but preferably not exceeding 28xc2x0 C. at a pH of 7 to 7.5 in a culture medium as defined above for the development of promastigote forms.
When the use intended for these parasitic stages does not require both axenic and aseric conditions, without the presence of macromolecules, to be used, it is possible within the scope of the invention to carry out the culture in a purely axenic medium, therefore in the presence of serum and also in the presence of macromolecules. For example RPMI medium which has foetal calf serum added to it can be used.
The short-term promastigote forms thus obtained can be used in the inoculation stage mentioned above for the purposes of the differentiation into amastigotes.
The transformation is very rapid and total, for example for Leishmania, in 4 days, after 2 to 5 subcultures, according to the species.
The implementation of the provisions of the invention allows standardized and reproducible cultures of the various forms corresponding to the various parasitic stages to be obtained, which are free from any cellular contaminant and from macromolecules and capable of multiplying in vitro.
The provisions which precede have been described more particularly relative to the promastigote forms and the amastigote forms of Leishmania whether cutaneous, muco- cutaneous or also visceral leishmanias, but also apply to the parasitic stages of T. cruzi or other hematoprotozoa such as Plasmodium and Babesia.
These cultures can be kept for several months, even several years for many species, in particular for Leishmania, whether they are cutaneous, mucocutaneous or visceral, or for T. cruzi.
The parasitic stages are capable of undergoing long-term cultivation, that is to say over more than 50 passages in in vitro cultures, and short-term cultivation, that is to say recently transformed from promastigote forms or from amastigote forms (earlier form of the cycle) of less than 10 passages.
The invention also supplies the means for producing a complete parasitic cycle in vitro. It will advantageously be noted that this complete cycle can be carried out in less than 15 days.
An embodiment of a process, according to the invention, for the production of stages corresponding to the developmental cycle of a parasite such as Leishmania is characterized in that it is used under the axenic and aseric conditions defined above, in the absence of macromolecules and in that it comprises:.
inoculation of a suitable culture medium, as defined above, with short-term or long-term promastigote forms, so as to obtain the differentiation into amastigote forms,
recovery of the amastigote forms produced, their inoculation and their culture as indicated above so as to obtain the differentiation into promastigote forms,
the cycle being repeated in its entirety, or partially, if desired, and indefinitely.
According to another embodiment, the short-term forms are cultivated under purely axenic conditions, or axenic and aseric conditions in the presence of macromolecules.
Via the process of the invention, it is therefore possible to obtain in vitro the various parasitic stages much more quickly and easily than by the in vivo techniques currently used which involve experimental infections which are sometimes difficult to bring about. These amastigote or promastigote parasitic forms are free from any cellular contaminant and are capable of multiplying in vitro. Thus means are available which allow the abundant, and even unlimited, obtaining of the parasitic stages recently, or not, differentiated from the earlier stage, in particular of Leishmania and those of T. cruzi (epimastigotes, metacyclic and bloodstream trypomastigotes and amastigotes).
Also a subject of the invention is the parasitic forms of the developmental cycle of tissular protozoan, such as Leishmania or T. cruzi, as obtained by implementing the culture process defined above.
These forms are characterized in that
they are free from cellular contaminants, in particular tissular macrophage and plasmatic contaminants accompanying the intracellular parasitic forms isolated from cell cultures or from tissues of experimentally-infected animals, as well as any seric contaminant, while being endowed with infective power in vitro and in vivo as observed on the intracellular forms when these are habitually infective, and their morphological, biochemical and immunological characteristics,
they are presented in the form of a homogeneous population relative to the age in culture and the state of differentiation for a given stage of the developmental cycle, this population, originating from a standardized culture, being capable of multiplying in vitro in a continuous manner.
The amastigote forms are particularly preferred given that they correspond to the forms developed during infection in man or animals.
These amastigote forms are also characterized in that they possess an enzymatic activity, more particularly a peptidase activity which is qualitatively more complex than those of the promastigotes, and quantitatively different, more particularly at the level of the cysteine-protease activities, as set out in the examples.
The invention relates more particularly to the amastigote forms of both anthroponotic and zoonotic, or anthropozoonotic Leishmania.
They can be the amastigote forms of cutaneous or muco- cutaneous Leishmania. Amongst these, there can be mentioned L. mexicana, L. amazonensis, L. braziliensis, L. guyanensis and L. panamensis. They can also be the amastigotes of visceral leishmanias such as L. chagasi, L. donovani or L. infantum.
Other amastigote forms according to the invention are those of various clones of T. cruzi.
The invention also relates to the short-term promastigote forms as defined above.
They can be populations directly transformed from amastigote forms which have an infective power similar to that of promastigotes recently isolated from an infected host.
Each of the parasitic stages, promastigote or amastigote, has different growth phases during multiplication in vitro, namely a latent phase, and exponential phase and a stationary phase which correspond to the preparation of the division, to an intense multiplication, then to a non-division stage respectively.
The invention advantageously allows forms corresponding to one of these phases to be obtained in a targeted manner and their properties and specific biochemical characteristics to be studied.
These forms are characterized in that they are free from cellular and seric contaminants, as well as molecules non-dialyzable at a cut-off threshold of 3 kDa.
They are therefore populations defined according to a well-determined phase of their growth.
The corresponding amastigote or short-term promastigote forms are particularly preferred.
The invention also relates to the total polypeptide extracts of these parasitic forms as obtained by lysis of the cells and recovery of the soluble or insoluble products. These extracts are also called total antigenic extracts in the examples. By these expressions xe2x80x9ctotal polypeptide extractsxe2x80x9d or xe2x80x9ctotal antigenic extractsxe2x80x9d is meant the products as obtained by lysis of the parasitic forms, whether they are of protein, lipid or saccharide nature.
They can be in particular total polypeptide extracts of short-term promastigote forms and quite particularly total polypeptide extracts of amastigote forms at different stages of their growth in vitro.
These extracts are characterized by their peptide profile as revealed in a standard manner using SDS-PAGE polyacrylamide gel, under reducing condition or not, or on polyacrylamide gel under non-denaturing condition, as described in the examples for certain species of Leishmania.
The invention also relates to the antigenic, protein, glucide or lipid fractions and determinants eluted or isolated from fractions of these extracts.
The antigenic fractions and determinants of these extracts recognized, according to an antigen-antibody type reaction, by sera of animals immunized with the total polypeptide extracts or sera of natural or experimental infections are particularly preferred with regard to the immunological applications which are a subject of the invention, and in particular the basic specific antigenic fractions and determinants.
Products of this type correspond to the antigens expressed on the surface of the amastigote forms and to the somatic antigens present at the level of the flagellar pocket or of vacuolar type as revealed by immunofluorescence techniques.
The purified or semi-purified molecules and the solutions specifically enriched with one or more of these molecules also come within the scope of the invention, originating from natural lyses.
Other products which are of great interest with regard to the biological applications according to the invention correspond to the excretion antigens as obtained from the culture supernatants conditioned by the promastigote forms or by the amastigote forms cultivated under the axenic and aseric conditions of the invention, originating from natural lyses.
The same goes for the differentiation antigens secreted during differentiation according to the process of the invention of the promastigote forms into amastigote forms and that of, the amastigote forms into promastigote forms (during the in vitro production of the cycle).
These antigenic products are recovered from the supernatants by simple concentration and dialysis. These supernatants therefore constitute a pre-enriched purified source of antigenic products.
The invention also relates to the immunization sera as obtained after administration of the antigenic extracts, fractions and molecules defined above according to the usual techniques, and the antibodies recovered from these sera.
It also relates to the infection sera obtained by the infection of animals with the infectious amastigote forms.
The antibody content of these sera varies according to the phase of the parasitic stage and is higher against the stationary phase of the amastigote forms.
The antibodies of the invention are characterized in that they recognize the specific peptides of amastigote or promastigote parasitic forms by producing an antigen-antibody type reaction.
Such antibodies include those only specifically recognizing the antigens of an amastigote or promastigote parasitic form, belonging to a homologous species, that is to say to the same species as that used for obtaining them, the recognized form being that against which they have been formed.
They can be for example antibodies formed against an amastigote form of Leishmania of a given species and which only specifically recognize the antigenic peptides of the amastigote forms of this species of Leishmania.
The antibodies of the invention also include those which in addition have a poor recognition of the other parasitic form of the species considered.
There can be mentioned for example the anti-amastigote antibodies recognizing, to a lesser degree, antigens of the promastigote forms of the same species.
Other antibodies also of the invention are in addition capable of recognizing, but to a lesser degree, the parasitic forms of a heterologous species, or of another genus such as T. cruzi. Antibodies of this type correspond for example to anti-amastigote antibodies of a species of Leishmania recognizing amastigote peptides of another species of Leishmania.
According to another aspect, the invention relates to the monoclonal antibodies as advantageously obtained according to the standard techniques of fusion of a cell line with the spleen or ganglion lymphocytes of an animal immunized by injection with a total peptide extract, an antigenic fraction or molecule as defined above by screening the supernatants of the hybridomas obtained, for example according to the Elisa or IF technique so as to reveal the antibodies directed specifically against a parasitic form of a species, as well as the clones of hybridomas secreting these monoclonal antibodies.
These antibodies constitute tools for selectively separating or isolating specific antigens of species or of stages from a medium containing them and in particular from the total polypeptide extracts mentioned above by immuno-affinity techniques.
The reaction of the above immunosera with antigenic fractions and molecules originating from a given phase of the development of the parasitic form allows the identification and isolation of the specific antigens of this stage.
The possibility of the mass production, due to the invention, of the cultured amastigote forms makes it possible to extract the total and messenger RNA""s and, from these, to create a cDNA library.
According to another aspect, the invention therefore relates to the total RNA""s as recovered from parasitic cultures of amastigotes or promastigotes, and the corresponding m-RNA""s and cDNA""s.
By comparison with a cDNA library of corresponding promastigote forms, specific peptides of a given parasitic stage are then revealed and their synthesis is proceeded with if appropriate by genetic engineering.
The obtaining, in accordance with the invention, of in vitro and in vivo infecting extracellular amastigote forms, having morphological, biological and biochemical characteristics similar to those of the intracellular amastigote forms, opens up new and numerous applications in the domains of research and industry.
The parasitic forms of the invention are thus particularly useful as experimental models for carrying out a first, screening in vitro of products which can be active for them in vivo.
The screening process of the invention comprises:
putting the parasitic forms, more especially the amastigote forms as cultivated under axenic conditions and notably aseric conditions, and the promastigote forms as cultivated in a completely defined medium, in contact with the products to be tested,
incorporation of nucleotides or amino acids labelled with a radioactive group, for example tritiated thymidine, in order to determine the activity of the products to be tested, or the carrying out of viability tests using for example a tetrazolium salt such as MTT.
In the stage of putting in contact, the parasitic forms are used at concentrations of 106 parasite/ml and the activity of the medicaments is studied at increasing concentrations.
The products to be tested can advantageously be labelled, for example with a radioactive group, to determine the action mechanisms and the flow of the drugs.
Thus the invention provides a model allowing, if desired, the comparison of the in vitro activity of medicaments on the promastigote form and on the amastigote form at different phases of their growth for a given parasite, and the testing of this activity on the actual form which is found in the host.
It allows improved characterization of the medicamentous activity by revealing either a lytic effect (leishmanicidal or trypanocidal), or an inhibitory effect on the multiplication (leishmaniostatic or trypanostatic) of the product.
The use of these parasitic forms as experimental models also allows the chemical resistance of the parasitoses to be studied.
In fact it is known that at present the resistance to medicaments constitutes a significant problem. Study of the mechanisms producing this resistance, which can easily be achieved using the models of the invention, is therefore of great interest.
The invention also relates to a kit for screening products which can be used for the treatment of parasitoses, more especially leishmanias or Chagas"" disease.
This kit comprises a support such as a multi-well plate containing the parasitic forms on which is it desired to test the activity of the product to be studied, some of these forms being used as controls, and reagents to determine the medicamentous activity of the product on the parasitic forms.
As indicated above, studies carried out up to now have not allowed parasitoses to be satisfactorily identified.
In man, or in animals and particularly in dogs, the diagnosis of leishmaniasis for example is most often determined either by isolating the parasite and identifying it (parasitological examination), or by detecting (specific circulating) antibodies in the serum (immunoserological tests).
The industrial culture of the amastigote forms under axenic or aseric conditions and in the absence of macromolecules makes available an abundant source of useful diagnostic tools.
In this way they allow the early detection, with a high degree of sensitivity and a high specificity, of circulating antibodies directed against the parasites.
The invention therefore relates to a method for the diagnosis of a parasitosis in man or animals, more especially of an infection caused by Leishmania or T. cruzi, or for their detection and identification in the vector insect, characterized in that it comprises:
putting a biological sample originating from the patient or the animal to be examined in contact with an amastigote form from an axenic and aseric culture or a promastigote form from an aseric culture as defined above, or a total polypeptide extract of these amastigote or promastigote forms, or one or more antigens specific for this extract, purified or semi-purified,
detection of the immunological complex.
The biological sample is more particularly a biological fluid such as blood, serum or urine.
When a purified or semi-purified, of a whole parasite antigen is used, it is immobilized on a support.
Latex beads, Elisa plates or fluorescence slides are for example used.
The reaction can be revealed directly by macroscopic agglutination in the direct or indirect agglutination test by reacting a conjugated antibody with fluorescein (indirect immunofluorescence technique) or with an enzyme such as peroxidase or alkaline phosphatase (ELISA tests).
A positive reaction therefore allows the presence of antibodies circulating in the patient or animal examined to be diagnosed.
The invention also relates to a kit for implementing a method for diagnosing a parasitosis, as defined above, in man or in animals.
This kit is characterized in that it comprises:
the antigenic reagents in immobilized form, namely the amastigote forms from axenic culture or axenic and aseric culture or the promastigote forms of defined culture, the total polypeptide extracts obtained from these forms or the antigens specific for these extracts with, if appropriate,
a positive control, constituted by a serum of known titer,
a negative control, as well as
the buffers and reagents which can be used for revealing the immunological reaction.
The detection of circulating anti-parasite antibodies in a patient or an animal can be carried out by putting in competition with the antibody of the sample, a specific antibody of the antigen, in particular a monoclonal antibody. The antibody advantageously contains a label, for example a radioactive or enzymatic group.
As a variant, the diagnostic method is based on revealing the presence of the antigenic determinants of the parasitic forms (detection of circulating antigens).
In this variant, the biological sample originating from the man or animal is put in contact with specific antibodies directed against the antigens of the parasitic form, or fragments of these antibodies.
The detection of the immunological reaction is carried out for example using the same antibody but labelled.
It is useful to note that the possibility provided by the invention of detecting circulating antigens, that is to say those which appear rapidly in the infected patient, allows early diagnosis of the disease to be carried out.
Antibodies directed against the promastigote or amastigote forms are used, in particular monoclonal antibodies, these forms originating from parasites of different species of leishmanias such as L. infantum or T. cruzi.
A corresponding diagnostic kit comprises:
an appropriate solid phase serving as support for the immunological reaction, such as a microtitration plate,
a preparation of antibodies according to the invention as defined above, or of fragments of these antibodies, immobilized on a support,
a positive control, constituted by a serum of known titer,
a negative control, as well as
the buffers and reagents which can be used to carry out the immunological reaction and in particular the labelled homologous antibody.
According to another aspect, the diagnostic tools of the invention allow a differential diagnosis to be carried out between several parasitoses.
In fact both in man and in animals, for example rodents, cross reactions between T. cruzi, T. rangeli (trypanosome non-pathogenic to man) and visceral or cutaneous leishmanias are observed.
The study of the parasitic forms of the invention, of the total polypeptide extracts and of the specific antigens defined above has revealed their strong immunogenic properties.
The invention therefore also relates to their use as protective agents vis-à-vis parasitoses, more especially leishmaniases and Chagas"" disease.
The vaccine compositions of the invention are characterized in that they are developed from amastigote forms or promastigote forms from axenic and aseric culture, in the absence of macromolecules, as defined above, at different phases of their growth, or from their constituents, in combination with a vehicle and/or an administration adjuvant.
The constituents of the amastigote or promastigote forms in question include the total polypeptide extracts obtained by lysis of these parasitic forms. They also include the antigenic fractions and the specific protective antigens isolated from the parasitic forms, but also from the culture supernatants conditioned by the parasites when they are cultivated in completely defined media.
The administration of these protection agents to man or animals allows them to be given an overall immunity against the parasitoses in which they occur in the natural infection process.
Their advantageous effect was especially revealed at the level of the immune response to cell mediation favouring the stimulation of the T lymphocytes secreting interleukin 2, and gamma interferon (TH1) or inhibiting the activation of the T cells secreting interleukins 4 and 5 (TH2).
These protection agents are advantageously in lyophilized form.
In the case of total polypeptide extracts, the vaccine compositions are administered by subcutaneous route at a rate of 100 to 1000 xcexcg in man and 100 to 500 xcexcg in dogs in the presence of adjuvants such as muramyldipeptide or saponin or in the presence of cytokine such as gamma interferon.
The excretion-secretion antigens of the culture supernatants metabolized by the amastigote forms of the invention offer an original strategy in the development of vaccines against parasitic diseases.
Their use for producing vaccines against canine or human visceral leishmaniases (L. intantum and L. chagasi) can in particular be emphasized. In fact they advantageously correspond to the forms present in the infected host.
The various experiments carried out have allowed their immunogenic properties and their protective effect for man or animals to be revealed.
To prepare vaccines from the said antigens, or ectoantigens, one uses the dialyzed concentrated supernatants of amastigote cultures, or the cultures themselves containing the parasites and the supernatants, the parasites being killed for example by thermal treatment, or extracts or these solutions.
These products are used with adjuvants such as MDP or cytokines, such as gamma interferon.
A long-term or short-term immunization protocol can be used. The long-term protocol is for example carried out by injection of the vaccine preparation every three weeks, on days d=0, d=21 and d=42. For a short-term protocol, for example, two injections are given with a two week interval.
After the virulence test, for animals, it is verified that the hypersensitivity immunity to cell mediation has indeed been induced towards the activation of the TH1 cells secreting interleukin 2 and gamma interferon.
These analyses can also be carried out for man at the end of the immunization.
Other characteristics and advantages of the invention are reported in the examples which follow.

Process for the in vitro culture of different stages of tissue parasites

Institut francais de recherche scientifique pour le developpement en Cooperation (ORSTOM)